Figure 5.
Figure 5. NK cell serial killing of target cells expressing different ligands is determined by the order of engagement. (A–J) NK cells were incubated with both Daudi-rituximab and Daudi-MICA at an E:T:T of 1:1.5:1.5 in microwells. Images were captured every 3 min for 8 h. (A) Representative image of one well (450 × 450 µm2) in fluorescence overlaid onto brightfield. NK cells are shown in cyan, Daudi-rituximab (Rtx) in blue, Daudi-MICA in green, and dead cells in red. Bar, 100 µm. (B) Representative images of enlarged regions from one microwell with time indicated. Bar, 20 µm. The example sequence shows how an NK cell (cyan) contacted a Daudi-rituximab (blue, 15 min) and killed it (36 min; yellow arrow). The NK cell detached, encountered a Daudi-MICA (green, 156 min) and killed it (168 min; target cell loses green fluorescence and begins to turn red; white arrow). Both targets were dead (red), whereas the NK cell remained alive (234 min). (C) Time required to lyse Daudi-rituximab and Daudi-MICA from initial contact (median ± IQR). (D) Time for target cell lysis stratifying initial and successive kills (median ± IQR). (E) Percentage of target cell lysis when Daudi-rituximab was encountered after contact with another Daudi-rituximab or Daudi-MICA. (F) Percentage of Daudi-MICA lysis when Daudi-MICA was met after Daudi-rituximab or Daudi-MICA. (G–J) Time-lapse microscopy was analyzed to record the outcome of different combinations of sequential interactions. Either no targets were killed (No kill), only the first (1st) or only the second (2nd) target was killed, or both were killed. Interactions that were unclear, e.g., many cells clustered together, were excluded from analysis. Graphs show the percentage of target cells killed from the following sequential encounters; (G) Daudi-rituximab then Daudi-rituximab, (H) Daudi-MICA then Daudi-MICA, (I) Daudi-rituximab then Daudi-MICA, and (J) Daudi-MICA then Daudi-rituximab. n = 231 interactions from three independent experiments; mean ± SD. *, P < 0.05; **, P < 0.01; ***, P < 0.001 by Mann Whitney test (C), Kruskal-Wallis test (D), Student's t test (E and F), or one-way ANOVA (G–J).

NK cell serial killing of target cells expressing different ligands is determined by the order of engagement. (A–J) NK cells were incubated with both Daudi-rituximab and Daudi-MICA at an E:T:T of 1:1.5:1.5 in microwells. Images were captured every 3 min for 8 h. (A) Representative image of one well (450 × 450 µm2) in fluorescence overlaid onto brightfield. NK cells are shown in cyan, Daudi-rituximab (Rtx) in blue, Daudi-MICA in green, and dead cells in red. Bar, 100 µm. (B) Representative images of enlarged regions from one microwell with time indicated. Bar, 20 µm. The example sequence shows how an NK cell (cyan) contacted a Daudi-rituximab (blue, 15 min) and killed it (36 min; yellow arrow). The NK cell detached, encountered a Daudi-MICA (green, 156 min) and killed it (168 min; target cell loses green fluorescence and begins to turn red; white arrow). Both targets were dead (red), whereas the NK cell remained alive (234 min). (C) Time required to lyse Daudi-rituximab and Daudi-MICA from initial contact (median ± IQR). (D) Time for target cell lysis stratifying initial and successive kills (median ± IQR). (E) Percentage of target cell lysis when Daudi-rituximab was encountered after contact with another Daudi-rituximab or Daudi-MICA. (F) Percentage of Daudi-MICA lysis when Daudi-MICA was met after Daudi-rituximab or Daudi-MICA. (G–J) Time-lapse microscopy was analyzed to record the outcome of different combinations of sequential interactions. Either no targets were killed (No kill), only the first (1st) or only the second (2nd) target was killed, or both were killed. Interactions that were unclear, e.g., many cells clustered together, were excluded from analysis. Graphs show the percentage of target cells killed from the following sequential encounters; (G) Daudi-rituximab then Daudi-rituximab, (H) Daudi-MICA then Daudi-MICA, (I) Daudi-rituximab then Daudi-MICA, and (J) Daudi-MICA then Daudi-rituximab. n = 231 interactions from three independent experiments; mean ± SD. *, P < 0.05; **, P < 0.01; ***, P < 0.001 by Mann Whitney test (C), Kruskal-Wallis test (D), Student's t test (E and F), or one-way ANOVA (G–J).

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